Irisin Prevents the Effects of Simulated Microgravity on Bone and Muscle Differentiation Markers

Abstract

Microgravity exposure affects both tissues and cells, and, in this regard, one of the most affected targets is the skeletal muscle system due to the significant loss of bone and muscle mass leading to osteoporosis and sarcopenia, respectively. Several efforts are underway to counteract the effects of microgravity, and recent studies on irisin, a myokine with anabolic effects on the musculoskeletal system, have shown promising results. Due to the practical challenges of conducting experiments in actual microgravity, different devices generating a simulated microgravity condition on Earth have been developed. Here, we exposed myoblasts, osteoblasts, osteocytes to a random position machine (RPM) for five days to assess microgravity effect on the expression of key differentiation factors in cells untreated or treated with irisin. In myoblasts (C2C12), exposure to RPM led to increased expression of early myogenesis maker genes Pax7 (p = 0.0016), Myf5 (p = 0.0005) and MyoD (p = 0.0009). Irisin treatment in the last 8 h of RPM cultures prevented these increases by returning Pax7 (p = 0.0008) and MyoD (p = 0.01) to control values, and only partially Myf5. In bone cells, exposure to RPM for 5 days showed no effect in osteoblasts (MC3T3) but decreased the expression of Pdpn (p = 0.0285) and Dmp-1 (p = 0.0423) genes in osteocytes (MLO-Y4). Irisin treatment completely prevented the decline in Pdpn (p = 0.293) and Dmp-1 (p = 0.0339) levels. Overall, our data showed that the impact of RPM exposure keeps myoblasts and osteocytes in a proliferative state, and irisin treatment restores them to their baseline biological condition, suggesting that irisin can counteract the changes induced by simulated microgravity.

Keywords: bone; irisin; microgravity; muscle; myoblasts; osteoblasts; osteocytes; random position machine.

FIGURE 1

Representative images of myoblasts (C2C12), under different culture conditions. (a) myoblasts cultured in ground control conditions; (b) myoblasts exposed to simulated microgravity for 5 days; (c) myoblasts exposed to simulated microgravity for 5 days and treated with irisin during the last 8 h of RPM exposure (irisin‐RPM).

FIGURE 2

qPCR shows mRNA expression levels of Pax7, Myf5 and MyoD in myoblasts (C2C12) cells. Data indicate that irisin treatment prevents the increase of the expression of Pax7 (a) and MyoD (c) and brings the expression to values of control condition, while a partial recovery is observed in Myf5 (b) expression. Ctrl, control condition; RPM, random position machine.

FIGURE 3

qPCR shows mRNA expression levels of Adam10 and Fndc5 in myoblasts (C2C12) cells. (a) irisin treatment increases the gene expression of Adam10 in RPM condition compared with untreated RPM cell cultures and Ctrl cell cultures. (b) irisin treatment increases the gene expression of Fndc5 in RPM condition compared with control condition. Ctrl, control condition; RPM, random position machine.

FIGURE 4

Representative images of osteoblasts (MC3T3), under different culture conditions. (a) osteoblasts cultured in ground control conditions; (b) osteoblasts exposed to simulated microgravity for 5 days; (c) osteoblasts exposed to simulated microgravity for 5 days and treated with irisin during the last 8 h of RPM exposure (irisin‐RPM).

FIGURE 5

Representative images of osteocytes (MLO‐Y4), under different culture conditions. (a) osteocytes cultured in ground control conditions; (b) ostecytes exposed to simulated microgravity for 5 days; (c) osteocytes exposed to simulated microgravity for 5 days and treated with irisin during the last 8 h of RPM exposure (irisin‐RPM).

FIGURE 6

qPCR shows mRNA expression levels of Prx1, Runx2 and Opg in osteoblasts (MC3T3) cells. RPM condition does not affect the expression of Prx1 (a), Runx2 (b) and Opg (c). Irisin treatment of osteoblasts in RPM condition increases gene expression of Opg vs. Ctrl condition and RPM condition (c). Ctrl, control condition; RPM, random position machine.

FIGURE 7

qPCR shows mRNA expression levels of Pdpn, Dmp‐1 and Mepe in osteocytes cells (MLO‐Y4). Irisin treatment prevents the decrease in expression observed in RPM conditions for the Pdpn (a) and Dmp‐1 (b) genes and restores gene expression to Ctrl conditions. Exposure to RPM condition does not affect Mepe expression (c). Ctrl, control condition; RPM, random position machine.

FIGURE 8

qPCR shows mRNA expression levels of senescence and apoptosis markers in bone and muscle cells. In myoblasts RPM does not alter p53 (a), Bcl2 (c) and Bax (d) expression, while a decrease of p21 expression is observed (b). Irisin treatment decreases Bax (d) gene expression. In osteoblasts, RPM does not affect gene expression of senescence and apoptosis markers (e,f,g,h). In osteocytes, irisin treatment prevents the drop in p53 expression (i) observed after RPM exposure and restores values to control values. Moreover, RPM condition decreased p21 expression both in untreated and irisin group (j). No effect of RPM is observed on Bcl2 (k) and Bax (l) gene expression. Ctrl, control condition; RPM, random position machine.